The present invention relates to a method of producing controlled release implants adapted for the administration of bioactive recombinant growth hormones at a controlled and continuous rate to a host. More particularly, the invention relates to a method of purifying bovine growth hormone and porcine growth hormone produced by DNA technology in a form suitable for use in controlled release devices.
Growth hormones are proteins that are involved in regulating protein metabolism as well as fat, carbohydrate, and mineral metabolism. Growth hormones affect the metabolic processes of the body by increasing the rate of cellular protein synthesis and decreasing protein degradation, as well as by increasing the rate of use of fatty acids and decreasing the rate of use of carbohydrates for production of energy in the body.
Bovine growth hormone (BGH) and porcine growth hormone (PGH) are proteins containing 191 amino acid residues. These proteins are synthesized in the anterior pituitary gland as "pre-growth hormones" having 26 additional amino acid residues attached at the amino terminal end. These 26-amino acid residue sequences are cleaved off prior to secretion from the pituitary cells, yielding the mature hormones. Field trials using BGH purified from pituitary glands demonstrated increased milk production and improved feed-to-milk conversion in cows to which the hormone was administered (Machlin, L. J., Journal of Dairy Science, 56:575-580 [1973]). The potential economic value of this hormone sparked interest in obtaining BGH in commercial quantities at reasonable cost. Field trials of native PGH have shown increased growth rates in young swine receiving the hormone.
Thus, much work in recent years has focused on obtaining microbial synthesis of these commercially valuable hormones using recombinant DNA technology. Gene cloning and manipulation techniques, well known in the art, have been used to produce recombinant expression vectors capable of directing the synthesis of BGH and PGH. For example, microorganisms transformed with BGH-encoding cDNA linked to a regulatory system have been shown to produce the desired hormone. Keshet et al., (Nucleic Acids Research, 9:19-30 [1981]) reported the cloning and low level expression in E. coli of a full length BGH polypeptide as a fusion protein with a portion of pBR322-encoded .beta.-lactamase. In European Patent Application Publication No. 0 103 395, construction of several expression vectors, including vectors encoding BGH polypeptides with varying portions of the aminoterminal end deleted, is described. BGH polypeptides with varying portions of the amino-terminal end of the mature hormone deleted were found to retain biological activity and to be expressed at much higher levels than was the complete hormone in the expression systems described.
Administration of BGH to cattle and PGH to swine has hitherto been only marginally successful. Methods of delivery of drugs that are well known in the art include oral, nasal, rectal, topical, and parenteral injection routes of administration. However, it is inconvenient to administer drugs to cattle and swine by these methods because of the large expense and amount of time required to deliver the drug to each member of a large group of animals on a daily basis.
Subcutaneous implants provide an alternative means for administering sustained, effective dosages of recombinant BGH and PGH to each animal. The implant contains a hormone reservoir surrounded by a protective wall permeable to the hormone. The advantage of these delivery systems is that they provide for controlled and predictable release rates of the hormones to the animals over an extended period of time. Unfortunately, we have found that controlled release devices containing BGH and PGH produced by recombinant microorganisms in fermentation media are subject to swelling and partial disintegration after implantation. This phenomenon dilutes the hormone in the implant and adversely affects the rate of release of the hormone. Therefore, the commercial need for a method of producing recombinant growth hormones in a form capable of effectively being incorporated into a controlled release implant persists.